This proposal, in response to RFA AI-07-003, is for the development of a future generation smallpox vaccine that can be safely administered to a diverse population. We have made great strides in the proof-of-principle selection of appropriate protein targets and the initial formulation of a protein-based smallpox vaccine. However, in order to bring the vaccine to the next level, optimization of the vaccine formulation and further improvements of the vaccine are required. To accomplish this, we bring together investigators with different areas of expertise and develop collaborations between academic researchers from different disciplines and with industry. Our hypothesis is that a protein-based smallpox vaccine can more safely elicit a protective response in a broad range of recipients than the current FDA approved live smallpox vaccine. While we have shown that this approach is feasible, there are a number of steps that are needed to bring this approach to the next level. The majority of our prior development of a protein-based smallpox vaccine has been with Vaccinia virus proteins, which based on their high homology to variola virus proteins, could confer cross protection against smallpox. There is theoretical and experimental evidence that variola proteins may ultimately be better candidates to generate protection against smallpox and therefore we will produce the variola virus homologs of A33, B5, and L1. Additionally, the formulation of the proteins with adjuvant needs to be optimized. An iterative and sequential process is now required to bring the vaccine to the next level. To accomplish this we will: Aim 1. Produce the variola virus protein homologs of Vaccinia virus A33, B5, and L1 proteins (C-PERL) Aim 2. Optimize the protein/adjuvant vaccine formulation (U. Kansas) Aim 3. Evaluate, in a checkerboard fashion, the ability of the optimized vaccine formulation to generate immune responses and protect from Vaccinia virus challenge in mice (U. Penn) Aim 4. Examine immune responses and protection from monkeypox challenge in a non-human primate model (Southern Research Institute)